Bertrand Pourrut

Lead-induced genotoxicity to Vicia faba L.roots in relation with metal cell uptake and initial speciation

Formation of organometallic complexes in soil solution strongly influence metals phytoavailability. However, only few studies deal with the influence of metal speciation both on plant uptake and genotoxicity. In the present study, Vicia faba seedlings were exposed for 6h in controlled hydroponic conditions to 5 μM of lead nitrate alone and chelated to varying degrees by different organic ligands. Ethylenediaminetetraacetic acid and citric acid were, respectively, chosen as models of humic substances and low weight organic acids present in natural soil solutions. Visual Minteq software was used to estimate free lead cations concentration and ultimately to design the experimental layout. For all experimental conditions, both micronucleus test and measure of lead uptake by plants were finally performed. Chelation of Pb by EDTA, a strong chelator, dose-dependently increased the uptake in V. faba roots while its genotoxicity was significantly reduced, suggesting a protective role of EDTA. A weak correlation was observed between total lead concentration absorbed by roots and genotoxicity (r(2)=0.65). In contrast, a strong relationship (r(2)=0.93) exists between Pb(2+) concentration in exposure media and genotoxicity in the experiment performed with EDTA. Citric acid induced labile organometallic complexes did not demonstrate any significant changes in lead genotoxicity or uptake. These results demonstrate that metal speciation knowledge could improve the interpretation of V. faba genotoxicity test performed to test soil quality.

Heavy-Metal-Induced Reactive Oxygen Species: Phytotoxicity and Physicochemical Changes in Plants

As a result of the industrial revolution, anthropogenic activities have enhanced there distribution of many toxic heavy metals from the earths crust to different environmental compartments. Environmental pollution by toxic heavy metals is increasing worldwide, and poses a rising threat to both the environment and to human health.Plants are exposed to heavy metals from various sources: mining and refining of ores, fertilizer and pesticide applications, battery chemicals, disposal of solid wastes(including sewage sludge), irrigation with wastewater, vehicular exhaust emissions and adjacent industrial activity.Heavy metals induce various morphological, physiological, and biochemical dysfunctions in plants, either directly or indirectly, and cause various damaging effects. The most frequently documented and earliest consequence of heavy metal toxicity in plants cells is the overproduction of ROS. Unlike redox-active metals such as iron and copper, heavy metals (e.g, Pb, Cd, Ni, AI, Mn and Zn) cannot generate ROS directly by participating in biological redox reactions such as Haber Weiss/Fenton reactions. However, these metals induce ROS generation via different indirect mechanisms, such as stimulating the activity of NADPH oxidases, displacing essential cations from specific binding sites of enzymes and inhibiting enzymatic activities from their affinity for -SH groups on the enzyme.Under normal conditions, ROS play several essential roles in regulating the expression of different genes. Reactive oxygen species control numerous processes like the cell cycle, plant growth, abiotic stress responses, systemic signalling, programmed cell death, pathogen defence and development. Enhanced generation of these species from heavy metal toxicity deteriorates the intrinsic antioxidant defense system of cells, and causes oxidative stress. Cells with oxidative stress display various chemical,biological and physiological toxic symptoms as a result of the interaction between ROS and biomolecules. Heavy-metal-induced ROS cause lipid peroxidation, membrane dismantling and damage to DNA, protein and carbohydrates. Plants have very well-organized defense systems, consisting of enzymatic and non-enzymatic antioxidation processes. The primary defense mechanism for heavy metal detoxification is the reduced absorption of these metals into plants or their sequestration in root cells.Secondary heavy metal tolerance mechanisms include activation of antioxidant enzymes and the binding of heavy metals by phytochelatins, glutathione and amino acids. These defense systems work in combination to manage the cascades of oxidative stress and to defend plant cells from the toxic effects of ROS.In this review, we summarized the biochemiCal processes involved in the over production of ROS as an aftermath to heavy metal exposure. We also described the ROS scavenging process that is associated with the antioxidant defense machinery.Despite considerable progress in understanding the biochemistry of ROS overproduction and scavenging, we still lack in-depth studies on the parameters associated with heavy metal exclusion and tolerance capacity of plants. For example, data about the role of glutathione-glutaredoxin-thioredoxin system in ROS detoxification in plant cells are scarce. Moreover, how ROS mediate glutathionylation (redox signalling)is still not completely understood. Similarly, induction of glutathione and phytochelatins under oxidative stress is very well reported, but it is still unexplained that some studied compounds are not involved in the detoxification mechanisms. Moreover,although the role of metal transporters and gene expression is well established for a few metals and plants, much more research is needed. Eventually, when results for more metals and plants are available, the mechanism of the biochemical and genetic basis of heavy metal detoxification in plants will be better understood. Moreover, by using recently developed genetic and biotechnological tools it may be possible to produce plants that have traits desirable for imparting heavy metal tolerance.

Lead-induced DNA damage in Vicia faba root cells: Potential involvement of oxidative stress

Genotoxic effects of lead (0-20μM) were investigated in whole-plant roots of Vicia faba L., grown hydroponically under controlled conditions. Lead-induced DNA damage in V. faba roots was evaluated by use of the comet assay, which allowed the detection of DNA strand-breakage and with the V. faba micronucleus test, which revealed chromosome aberrations. The results clearly indicate that lead induced DNA fragmentation in a dose-dependant manner with a maximum effect at 10μM. In addition, at this concentration, DNA damage time-dependently increased until 12h. Then, a decrease in DNA damages was recorded. The significant induction of micronucleus formation also reinforced the genotoxic character of this metal. Direct interaction of lead with DNA was also evaluated with the a-cellular comet assay. The data showed that DNA breakages were not associated with a direct effect of lead on DNA. In order to investigate the relationship between lead genotoxicity and oxidative stress, V. faba were exposed to lead in the presence or absence of the antioxidant Vitamin E, or the NADPH-oxidase inhibitor dephenylene iodonium (DPI). The total inhibition of the genotoxic effects of lead (DNA breakage and micronucleus formation) by these compounds reveals the major role of reactive oxygen species (ROS) in the genotoxicity of lead. These results highlight, for the first time in vivo and in whole-plant roots, the relationship between ROS, DNA strand-breaks and chromosome aberrations induced by lead.

Molecular Mechanisms Involved in Lead Uptake, Toxicity and Detoxification in Higher Plants

Lead (Pb) is one of the most useful metals, and has contaminated most of the ecosystems. It has been known to influence various morphological, physiological and biochemical processes in plants. Higher plants have evolved sophisticated internal detoxification mechanisms, to deal with metal toxicity, that include selective metal uptake, excretion, chelation by specific ligands and compartmentalization. Despite these detoxification systems, Pb is able to induce the production of reactive oxygen species (ROS) which can cause harmful effects on vital constituents of plant cells: protein oxidation, lipid peroxidation, enzyme inactivation or DNA damages. Unlike other metals, little is known about the mechanisms involved in Pb uptake, toxicity, oxidative stress generation and detoxification in plants. This chapter reviews the recent understanding of physiological and biochemical mechanisms involved in Pb uptake and translocation into plants. It also provides a broad overview of the most important mechanisms of Pb toxicity and tolerance in plants.

Role of metal speciation in lead-induced oxidative stress to Vicia faba roots

Chemical speciation of metals in soil/solution plays an important role in determining their biogeochemical behavior in soil-plant system. The current study evaluated the influence of applied form of Pb (metal speciation) on its toxicity to metal sensitive Vicia faba L. roots. Lead was applied to young V. faba seedlings alone or chelated by organic ligands (citric acid and ethylenediaminetetraacetic acid). Plants were exposed to all treatments for 1, 4, 8, 12, and 24 h in nutrient solution, and contents of H2O2 and thiobarbituric-acid-reactive substances (TBARS) production were analyzed in V. faba roots. The results showed that Pb toxicity to V. faba roots depended on its applied chemical form and duration of exposure. Lead alone caused two burst of lipid peroxidation and H2O2 induction at 1 h and 12 h. Addition of EDTA dose-dependently inhibited Pb-induced H2O2 and TBARS production, indicating a protective role of this chelator against Pb toxicity during the first 24 h. In contrast, citric acid did not show significant effects on Pb-induced H2O2 and TBARS production, but delayed the induction of these effects. This study suggested that Pb toxicity to V. faba roots varies with Pb speciation in growth medium.

Recommendations for increasing alkaline comet assay reliability in plants

In plants, an increasing interest for the comet assay was shown in the last decade. This versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, the lack of a standardised protocol and the low throughput of the assay limit its use in plants. The aims of this paper are to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. We examined the effect of varying several parameters on four different plant species: broad bean (Vicia faba), white clover (Trifolium repens), English ryegrass (Lolium perenne) and miscanthus (Miscanthus x giganteus). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated. Results clearly indicate that short chopping is more efficient to isolate nuclei than the standard slicing method. Filtration and lysis steps were shown to be unnecessary and thus should be skipped. Data also demonstrate that high room temperatures and light could induce DNA damage in isolated nuclei. Calibration tests with H2O2 or ethyl methanesulfonate revealed that a special attention should be paid to plant growing stage, leaf position and exposure duration.